Methods for extracting and purifying kallidinogenase

ABSTRACT

A method for extracting and collecting kallidinogenase (EC 3.4.4.21 substance) which comprises extracting the pancreas of a mammal with water in the presence of a salt at a temperature of about 45° to about 60° C. and a pH of about 5 to about 8, wherein the extraction is carried out in the presence of a water-soluble salt of magnesium with an acid, and a method for purifying kallidinogenase which comprises subjecting the aqueous phase of a kallidinogenase-containing aqueous extract obtained from the pancreas of a mammal to an adsorption-elution treatment using an anion-exchange resin, wherein said aqueous phase of the kallidinogenase-containing aqueous extract is brought into contact with a gel-type weakly basic anion-exchange resin to cause the kallidinogenase fraction to be adsorbed to said resin, and the kallidinogenase is eluted with an aqueous solution of an ammonium salt of a weak acid, thereby to collect the kallidinogenase.

This is a division of application Ser. No. 638,042, filed Dec. 5, 1975,now U.S. Pat. No. 3,994,782.

This invention relates to an improved method for extracting andcollecting kallidinogenase (EC 3.4.4. 21 substance, known as Kallikreinby trademark) from the pancreas of mammal.

The method of this invention affords improved kallidinogenaseseparability (increased yield) whereby a kallidinogenase-containingaqueous phase with reduced amounts of protein and other impurities (lowturbidity) can be obtained in a greater amount that in prior arttechniques using the same amount of extracting water. Furthermore, thekallidinogenase-containing aqueous phase having high kallidinogenaseactivity can be obtained with operational advantages by a simple solidliquid separating procedure without requiring any means of separatingthe aqueous phase, for example, centrifugal separation. According tothis method, the kallidinogenase is very stable for heat under theextracting conditions of the invention as compared with the prior arttechniques, and advantageously exhibits no likelihood of denaturationduring the extracting step.

The present invention also relates to a method for purifyingkallidinogenase in which kininase can be removed very easily andsubstantially completely.

It has already been known to extract kallidinogenase, a circulatoryhormone, from the "pancreas of mammal" (as referred to in the presentapplication) such as the pancreas itself of a mammal, a powder of thepancreas, or a kallidinogenase-containing extract of the pancreas, andto purify the extracted crude kallidinogenase.

The conventional technique of extracting kallidinogenase from thepancreas of mammal comprises subjecting pancreas having a high fatcontent to a fat removing treatment which however requires a complicatedprocedure, and then extracting the defatted pancreas with water at anelevated temperature at a pH of about 4 to about 7. When thefat-removing treatment is omitted in this technique, the extractingoperation and the operation of separating and collecting the aqueousphase are extremely difficult. The method also has the defect that evenafter performing the fat removing treatment, the extracting operationand the aqueous phase-separating operation are still difficult, and theyield of kallidinogenase is low.

In an attempt to overcome the difficulties of this technique, animproved method was suggested in which the above extracting operation iscarried out using an aqueous solution of an alkali metal salt such assodium chloride or an ammonium salt in the co-presence of an organicsolvent at a pH of 5.5 to 9.5 (see Japanese Patent Publication No.17041/73). This suggestion is an improvement over the method in which nosalts are used, but suffers from disadvantages arising from the use oforganic solvents. It is desired to be improved further in regard to theyield (activity), heat stability, and turbidity of kallidinogenaseduring the extraction step.

On the other hand, various suggestions have been made in regard to thepurification of a kallidinogenase-containing aqueous extract obtainedfrom the pancreas of mammal by adsorption-elution techniques. Forexample, two types of purifying methods have been suggested, one typerelying on the use of polysaccharide-type non-resinous materials which,for example, includes a method comprising allowing the crudekallidinogenase to be adsorbed to a weakly basic anion-exchangecellulose and separating it (Japanese Patent Publication No. 11697/62),and a method comprising adding a lead or zinc salt to an aqueoussolution containing kallidinogenase, allowing the resultingkallidinogenase precipitate to be adsorbed to a weakly basicanion-exchange cellulose or cross-linked dextran (SEPHADEX: tradename)and separating it (Japanese Laid-Open Patent Publication No. 56889/73),the other type relying on the use of ion-exchange resins which, forexample, includes a method comprising adding a protein precipitant to anextract of the gland tissue of the pancreas, allowing the resultingkallidinogenase to be adsorbed onto a macroporous strongly basicanion-exchange resin, and separating it (Japanese Laid-Open PatentPublication No. 103715/73).

The methods utilizing a polysaccharide-type non-resinous material, asadsorbent, have the disadvantage that the physical strength of theion-exhange material is not sufficient, and contamination bymicroorganisms is liable to occur, and thus are not feasible forcommercial operations. On the other hand, the other type has theadvantage of avoiding the above-mentioned troubles, but is stillunsatisfactory in regard, for example, to the elution of the desiredproduct and the removal of undesirable kininase.

We made extensive investigations in order to remove the defects of theconventional methods for extracting and collecting kallidinogenase andthe conventional methods for purifying the kallidinogenase extract. As aresult, we found that kallidinogenase can be separated and collectedwith superior heat stability, separability, turbidity and activity byextracting the pancreas of mammal with water in the presence of awater-soluble salt formed between magnesium, which is a metal of aperiod 3 of Group II of the periodic table, and an acid. As will beshown later by comparative experiments, it has been found that accordingto the method of this invention, better results can be achieved than inthe case of using the alkali metal salts or ammonium salts indicatedabove with regard to the prior art techniques, and that the improvementattained by this invention cannot be achieved even when salts ofcalcium, a metal of period 4 of Group II of the periodic table, oraluminum, a metal of Group III of the periodic table, are used.

Furthermore, we found that by utilizing a gel-type weakly basicanion-exchange resin, a greater part of the undesirable kininase can beremoved completely in a washing step after adsorption, and the adsorbedkallidinogenase can be eluted and recovered with good efficiency and atgood recovery ratios by a subsequent eluting step which is lesstime-consuming than in the prior art. It has also been found that thissuperior purification effect is not obtained by using a gel-typestrongly basic anion exchange resin or a macroporous weakly basicanion-exchange resin, but is a unique effect obtainable only with thegel-type weakly basic anion-exchange resin.

Accordingly, it is an object of this invention to provide a superiorimproved method for extracting and collecting kallidinogenase from thepancreas of mammal.

Another object of this invention is to provide a superior method forpurifying a kallidinogenase containing aqueous extract obtained from thepancreas of mammal thereby to obtain purified kallidinogenase.

Other objects and advantages of this invention will become apparent fromthe following description.

According to this invention, there is first provided a method forextracting and collecting kallidinogenase which comprises extracting thepancreas of mammal with water in the presence of salts at a temperatureof about 45° to about 65° C. and a pH of about 5 to about 8, wherein theextraction is carried out in the presence of a water-soluble salt formedbetween magnesium and an acid.

The pancreas of mammal used in this invention may be an attenuated orminced product of the raw pancreas of mammal such as pig, cow, horse orwhale, or an acetone-treated pancreas powder or a water-extract of thepancreas. In the present invention, it is not necessary to subject theraw pancreas to a complicated defatting treatment in advance, but theraw pancreas, attenuated to a desired form to alloy easy extraction, canbe used directly.

According to the method of this invention, the pancreas of mammal isextracted with the water-soluble magnesium salt of an acid. Theextraction is carried out at a temperature of about 45° to about 60° C.at a pH of about 5 to about 8 for a period of, say, about 30 minutes toabout 2 hours. When the pH is less than about 5 or more than about 8,the kallidinogenase is liable to be deactivated during the extractingoperation, or the separability of the aqueous phase becomes poor,thereby causing a reduction in the yield of kallidinogenase andmoreover, the separating operation becomes difficult. When theextracting temperature is less than about 45° C., the separability ofthe aqueous phase becomes poor, and long periods of time are requiredfor separation by, for example, allowing the system to stand. Thisresults in the deactivation of the kallidinogenase and its reducedyield. On the other hand, when the extracting temperature exceeds about60° C., the kallidinogenase is liable to be deactivated, and thistendency is likely to increase unless the time required for separatingthe kallidinogenase by allowing the system to stand is shortened.However, the shortening of the separating time makes the separationinsufficient, and causes a reduction in the yield of kallidinogenase.

The extraction in accordance with the method of this invention iscarried out in the presence of a water-soluble salt of magnesium with anacid, which has been added to the extraction system. The water-solublemagnesium salt of an acid may, for example, be salts formed betweenmagnesium and acids such as mineral acids, carbonic acid, and fattyacids containing 1 to 5 carbon atoms. Specific examples of thewater-soluble magnesium salts are magnesium carbonate, magnesiumchloride, magnesium nitrate, magnesium sulfate, magnesium formate,magnesium acetate, magnesium propionate, magnesium butyrate, magnesiumlactate, magnesium malate, magnesium oxalate, magnesium succinate, andmagnesium valerate.

The extracting method of this invention makes it possible to afford akallidinogenase with superior heat stability, separability, turbidityand kallidonogenase activity as compared with the case of using alkalimetal salts or ammonium salts as previously suggested and also in thecase of using similar salts of calcium (a metal of Group II of theperiodic table) or of aluminum (a metal of period 3, Group III of theperiodic table). These advantages of this invention can be demonstratedexperimentally as follows:

The experimental procedure comprises adding 500 ml. of water to 100 g ofminced raw pancreas (the essential pancreas content: about 50%) of pig,adjusting the pH of the mixture to 6, adding each of the various saltsshown in Table 1, allowing the mixture to stand for 1 hour at 55° C. toextract and collect kallidinogenase. The separability, turbidity andrelative kallidinogenase activity of the kallidinogenase-containingaqueous extract, and the heat stability of kallidinogenase aredetermined. The results are shown in Table 1.

                                      Table 1                                     __________________________________________________________________________                    Kallidinogenase-containing                                                    aqueous extract                                                                              Heat-stability                                                           Relative                                                                           of kallidino-                                             Concent-       kallidino-                                                                         genase                                                    ration                                                                             Separ-    genase                                                                             (relative                                                 of salts                                                                           ability                                                                           Turbidity                                                                           activity                                                                           residual                                       Salts      (M)  (%) (O.D.600)                                                                           (%)  activity, %)                                   __________________________________________________________________________         Not added                                                                           --   57  3.1   60   77                                                  ammonium -                                                                          chloride                                                                           0.03                                                                              65    1.7  68     70                                           Sodium                                                                        acetate                                                                             0.03 60  1.7   75   83                                                  Sodium                                                                        chloride                                                                            0.03 72  1.7   88   86                                             Compar-                                                                            Sodium                                                                   ison chloride                                                                            0.06 70  1.6   88   86                                                  Sodium                                                                        sulfate                                                                             0.03 60  1.7   70   75                                                  Aluminum                                                                      acetate                                                                             0.03 75  1.7   20   35                                                  Calcium                                                                       acetate                                                                             0.03 72  1.7   66   83                                                  Calcium                                                                       chloride                                                                            0.03 75  1.7   70   85                                                  Magnesium                                                                     carbonate                                                                           0.03 81  1.3   90   93                                                  Magnesium                                                                     chloride                                                                            0.03 82  1.2   92   93                                             Inven-                                                                             Magnesium                                                                tion sulfate                                                                             0.03 84  1.2   98   97                                                  Magnesium                                                                     acetate                                                                             0.03 85  1.1   100  100                                                 Magnesium                                                                     lactate                                                                             0.03 84  1.2   95   96                                                  Magnesium                                                                     propionate                                                                          0.03 83  1.2   97   98                                                  Magnesium                                                                     butyrate                                                                            0.03 83  1.2   94   95                                                  Magnesium                                                                     succinate                                                                           0.03 81  1.2   91   93                                             __________________________________________________________________________

The heat stability of kallidinogenase is that of purifiedkallidinogenase maintained at 55° C. and a pH of 6.0 for 60 minutes, andexpressed as a relative residual activity (%) with the residual activityof kallidinogenase obtained in the presence of magnesium acetate takenas 100%.

The separability (%) represents the percentage of the amount of theextract based on the total amount of the liquid treated. The turbidityof the extract is measured at 660 mμ(nm). The activity is the BAEE(benzoyl arginine ethyl ester) decomposing activity of the extractmeasured in the presence of a 0.02% soybean trypsin inhibitor The BAEEdecomposing activity of the extract obtained in the presence ofmagnesium acetate is taken as 100%, and the measured value is convertedon this basis. One BAEE unit is the amount of kallidinogenase requiredto decompose 1 μ of BAEE per minute when the kallidinogenase is causedto act on BAEE at 37° C.

The extract obtained in the presence of magnesium acetate is welldialyzed. The kallidinogenase unit (KU) of the dialyzate as measured bythe increase of blood flow method (see, Hiroshi Moriya, "Basic Lecturesin the Development of Medicines", Vol. 5, Pharmacological tests, page974, a Japanese-language publication published in 1971 by Chijin ShokanCompany, Tokyo) is 75,000 per kilogram of the raw pancreas.

The effect of salts used for extracting kallidinogenase from a powder ora water extract of pig pancreas has been found to be of the sametendency as in the case of using the raw pancreas described above.

Preferably, the amount of the water-soluble magnesium-acid salt is suchthat the concentration of this salt is at least about 0.02 mole, andusually about 0.02 to about 1 mole. A suitable concentration can bechosen within this range according to the type of the salt to be used.The salt can be used in higher concentrations, but no appreciableincrease in effect can be obtained with an increase in concentration.When the concentration of the salt is less than about 0.02 mole, thekallidinogenase extracting efficiency becomes poor, and the separabilityof the kallidinogenase-containing aqueous phase from undesiredcomponents is poor. Furthermore, this leads to an increase in the amountof protein dissolved in the aqueous extract. Accordingly, thepost-treatment of the aqueous extract becomes complicated, and the yieldof kallidinogenase tends to decrease.

According to a second aspect of this invention, there is provided amethod for purifying kallidinogenase which comprises subjecting akallidinogenase-containing aqueous extract obtained from the pancreas ofa mammal to an adsorption-elution treatment using an anion-exchangeresin, wherein said kallidinogenase-containing aqueous extract isbrought into contact with a gel-type weakly basic anion-exchange resinto cause the kallidinogenase fraction to be adsorbed to said resin, andthe kallidinogenase is eluted with an aqueous solution of an ammoniumsalt of a weak acid, thereby to collect the kallidinogenase.

This purifying method is especially suitable for obtaining purifiedkallidinogenase from the aqueous extract obtained by the method of thisinvention. It can also be applied with equally superior purifyingeffects to kallidinogenase-containing extracts obtained by any othermethods.

The aqueous kallidinogenase-containing extract obtained by theextracting method of this invention can also be purified by anadsorption-elution procedure using silica gel, other knownpolysaccharide non-resinous material, as an adsorbent, or otherion-exchange resins.

We found that with a macroporous strongly basic anion-exchange resin anda macroporous weakly basic anion-exchange resin, the elution ofkallidinogenase adsorbed thereto is slow, and for complete elution andseparation, a large quantity of solvent and a long period of time arerequired, and moreover, the impurities tend to accompany the separatedkallidinogenase; but that by using the gel-type weakly basicanion-exchange resin, the intended kallidinogenase adsorbed thereto canbe very rapidly eluted using an aqueous solution of an ammonium salt ofa weak acid as an eluting solvent, and can be easily separated from theimpurities.

According to the purifying method of this invention, thekallidinogenase-containing aqueous extract is passed through a column ofthe gel-type weakly basic anion-exchange resin to ensure contactingbetween the aqueous extract and the ion-exchange resin, and thereby toadsorb the kallidinogenase fraction to the ion exchange resin.Preferably, the adsorption operation is carried out at a pH of more thanabout 4 but below about 8, more preferably about 5 to about 7,especially preferably about 6± 0.5 . At pH values outside the specifiedrange, the kallidinogenase is deactivated, and at a pH of more thanabout 8, the amount of kallidinogenase not adsorbed increases.Consequently, the yield and purity of the kallidinogenase are reduced.

The kallidinogenase adsorbed to the gel-type weakly basic anion-exchangeresin is eluted using an aqueous solution of an ammonium salt of a weakacid at a pH of about 6 to about 7. The temperature at which theadsorption and elution are carried out may be any temperature at whichkallidinogenase does not decompose. Usually, these operations areperformed at room temperature. The salt can be removed from the eluateby such means as dialysis or ultrafiltration, and the resulting productcan be dried at a temperature below the decomposition temperature of thekallidinogenase, usually below about 60° C. The drying can be done, forexample, by lyophilizing or spray-drying techniques.

The gel-type weakly anion-exchange resin used in the performance of theabove purifying method is preferably a resin having a secondary ortertiary amine as an exchange group, examples of which aredivinylbenzene acrylate type resin (DIAION WA 10, DIAION WA 11,trademarks for products of Mitsubishi Chemical Industries Ltd., Japan),and styrene divinylbenzene type resins (AMBERLITE IR 45, AMBERLITE IRA47, AMBERLITE IRX 68, trademarks for products of Rohm & Haas Co.,U.S.A).

In order to demonstrate the effect of the gel-type weakly basicanion-exchange resin, the following experiment was performed usingDIAION WA 10 as the gel-type weakly basic anion-exchange resin inaccordance with this invention, and as other anion-exchange resins, amacroporous weakly basic anion-exchange resin (DIAION WA 30, trademarkfor a product of Mitsubishi Chemical Industries, Ltd.), a gel-typestrongly basic anion-exchange resin (DIAION SA 21A, trademark for aproduct of Mitsubishi Chemical Industries, Ltd.), and a Macroporousstrongly basic anion-exchange resin (DIAION HPA 10, trademark for aprodut of Mitsubishi Chemical Industries, Ltd.)

The experimental procedure comprises passing 300 ml. of an extract ofthe raw pancreas of a pig (3 BAEE units/ml. as kallidinogenase) througha column (3 × 25 cm) of each of the ion-exchange resins mentioned above,washing the resin with a 0.2M solution of ammonium acetate (pH 6.0), andeluting the extract using a 0.4M solution of ammonium acetate (pH 6.0)at a flow rate of 50 ml/hr while collecting the eluate in fractions eachwith a volume of 19 ml. per test tube. The results are shown in Table 2.

Referring to Table 2, the kallidinogenase is expressed by BAEE units.

The carboxypeptidase (CPase) is expressed by HPLA (hippuryl-L-β-phenyllactate) decompsing activity. One HPLA unit is the amount of CPaserequired to decompose lμ mole of HPLA per 1 minute when CPase is causedto act on HPLA. The measurement of the HPLA decomposing activity isemployed in this invention as a method for detecting a kininase fractionin chromatography.

                                      Table 2                                     __________________________________________________________________________                 Invention                                                                              Comparison 1                                                                           Comparison 2                                                                           Comparison 3                          __________________________________________________________________________    Ion exchange                                                                               DIAION WA 10                                                                           DIAION WA 30                                                                           DIAION SA 21A                                                                          DIAION HPA 10                         resin                                                                         Characteristics                                                                            weakly basic,                                                                          weakly basic,                                                                          strongly basic,                                                                        strongly                              of the ion   gel-type macroporous                                                                            gel-type basic,                                exchange resin                          macroporous                           Amount of the                                                                 extract of the                                                                             300 ml   300 ml   300 ml   300 ml                                big pancreas                                                                  __________________________________________________________________________    Enzymes      KA* Cpase                                                                              KA* CPase                                                                              KA* Cpase                                                                              KA* CPase                             Amount adsorbed                                                                            800 39000                                                                              900 107000                                                                             800 50000                                                                              900 88000                             Amount of the                                                                 enzymes eluted                                                                             0   38400                                                                              0   31200                                                                              0   15000                                                                              0   80000                             during washing                                                                             (0) (98) (0) (29) (0) (30) (0) (91)                              __________________________________________________________________________         1st fraction                                                                  1st to 16th                                                                           790 250  190 61360                                                                              92  890  420 3000                                   test tubes                                                                    (0-304 ml.)                                                                           (99)                                                                              (0.6)                                                                              (21)                                                                              (57) (11)                                                                              (2)  (47)                                                                              (3)                               Amount                                                                             2nd fraction                                                             of the                                                                             17th to 46th                                                                          0   0    405 14250                                                                              0   0    380 2000                              enzyme                                                                             test tubes                                                               eluted                                                                             (305-874 ml.)                                                                         (0) (0)  (45)                                                                              (13) (0) (0)  (42)                                                                              (2)                                    3rd fraction                                                                  47th to 96th                                                                          0   0    300 144  0   0    50  0                                      test tubes                                                                    (875-1,824 ml.)                                                                       (0) (0)  (35)                                                                              (0.1)                                                                              (0) (0)  (6) (0)                               __________________________________________________________________________     *KA stands for kallidino-genase.                                         

The figures in the parentheses represent the percentages of the amountof the eluted enzyme based on the amount of the enzyme adsorbed to theion exchange resin.

The biological activity of the kallidinogenase in those eluted fractionswhich had BAEE decomposing activity was ascertained by the increase ofblood flow method, and the biological activity of the kininase in thoseeluted fractions which had HPLA decomposiing activity was ascertained bya rat uterus smooth muscle shrinking method (see Hiroshi Moriya, "basicLectures in the Development of Medicines", Vol. 5, Pharmacological TestMethods, page 974, a Japanese-language publication published by ChijinShokan Company, Tokyo in 1971).

It can be seen from the results shown in Table 2 that according to thepurifying method of this invention using the gel-type weakly basicanion-exchange resin, about 99% of kallidinogenase was eluted andrecovered in the first fraction of 304 ml, and the eluting andrecovering efficiency was far superior to those obtained in the case ofusing the gel-type strongly basic anion-exchange resin, the macroporousstrongly basic anion-exchange resin, and the macroporous weakly basicanion-exchange resin. The results also demonstrate that the amount ofCPase, an undesirable impurity, is far smaller in the invention than inthe comparison runs; at the time of washing the resin before elution, agreater part (98%) of the adsorbed CPase can be removed; substantiallyall (99%) of the kallidinogenase can be eluted and recovered at theearly stage of elution; and that the amount of CPase is very small(0.6%), and the quality of the purified kallidinogenase obtained by onecycle of the adsorption-elution procedure has very good quality.

The following Examples illustrate the present invention in greaterdetail. In these examples, the essential pancreas contents were about50% by weight based on the pancreas used. The pancreas was used withoutremoving the fat.

The kininase activity is expressed by kininase units. One kininase unitis the amount of kininase required to decompose 1 ng of bradykinin perminute.

EXAMPLE 1

500 g of raw pancreas of pig was minced, and 2,000 ml. of water wasadded. The pH of the mixture was adjusted to 6.0. 12 g (concentration0.05M) of magnesium sulfate was added to the above mixture, and theywere well mixed. The mixture was allowed to stand at about 50° C. for1.5 hours. The extract separated as a bottom layer was taken out, andkallidinogenase was obtained in an amount corresponding to 36,000kallidinogenase units (KU).

EXAMPLE 2

500 g of raw pancreas of pig was minced, and 2,000 ml. of water wasadded. The pH of the mixture was adjusted to 6.0. 13 g (concentration0.03 M) of magnesium acetate was added to the mixture, and they werewell mixed. The mixture was allowed to stand at about 55° C. for 1 hour.The extract separated as a bottom layer was taken out, andkallidinogenase was obtained in an amount corresponding to 37,000 KU.

EXAMPLE 3

100 g of an acetone-dried powder of the pancreas of pig was mixed with3,000 ml. of water, and the pH of the mixture was adjusted to 6. 16 g(concentration 0.025 M) of magnesium acetate was added to the mixture,and they were well mixed. The mixture was allowed to stand at about 50°C. for 1.5 hours. The extract separated as a bottom layer was taken out,and kallidinogenase was obtained in an amount corresponding to 73,000KU.

EXAMPLE 4

3,000 ml. of water was added to 100 g of a water extract of the pancreasof pig, and the pH of the mixture was adjusted to 6. 18 g (concentration0.05 M) of magnesium sulfate was added to the mixture, and they werewell mixed. The mixture was allowed to stand at about 47° C. for 2hours. The extract separated as a bottom layer was taken out, andkallidinogenase was obtained in an amount corresponding to 72,000 KU.

EXAMPLE 5

300 g of raw pancreas of pig was minced, and 900 ml. of water was addedand the pH of the mixture was adjusted to 6. 6.25 g (concentration 0.03M) of Magnesium acetate was added to the mixture, and they were wellmixed. The mixture was allowed to stand at about 50° C. for 1.5 hours.The mixture was allowed to stand at room temperature to remove the toplayer containing fat. The bottom layer was subjected to centrifugalseparation to obtain 800 ml of the extract. The extract was passedthrough a column (3 × 25 cm) of a gel-type weakly basic anion-exchangeresin (DIAION WA 10) buffered with a 0.15 M solution of ammonium acetate(pH 6.0), and after washing the resin with a 0.2 M solution of ammoniumacetate (pH 6.0), the extract was eluted with a 0.4 M solution ofammonium acetate (pH 6.0).

The salt was removed from the eluate, and the resulting kallidinogenasefraction was lyophilized to obtain 148 mg (117.7 KU/mg) ofkallidinogenase. The kininase unit of this product per kallidinogenaseunit (KU) was only 0.5.

EXAMPLE 6

30 g of an acetone-dried powder of the pancreas was mixed well with 20times its amount of water and the pH of the mixture was adjusted to 6.3.2 gram (concentration 0.05 M) of magnesium sulfate was added to themixture, and they were well mixed. The mixture was allowed to stand atabout 45° C. for 2 hours. The mixture was then allowed to stand at roomtemperature, and the top layer containing solids such as insolubleprotein was removed. The bottom layer was subjected to centrifugalseparation to obtain 550 ml of the extract. The extract was passedthrough a column (3 × 25 cm) of a gel-type weakly basic anion-exchangeresin (AMBERLITE IRX 68) buffered with a 0.15 M solution of ammoniumacetate (pH 6.0), and after washing the resin with 600 ml. of a 0.2 Msolution of ammonium acetate (pH 6.0), the extract was eluted with a 0.4M solution of ammonium acetate (pH 6.0).

The salt was removed from the eluate, and the resulting kallidinogenasefraction was lyophilized to obtain 156 mg (142.3 KU/mg) ofkallidinogenase. The kininase unit of this product per kallidinogenaseunit (KU) was only 1.

EXAMPLE 7

The extracts obtained in Example 1 and Example 2 were each eluted anddried in the same was as in Example 5 to obtain 257 mg and 241 mg,respectively, of kallidinonase. The kallidinogenase activities were108.3 KU/mg and 126.2 KU/mg, respectively.

EXAMPLE 8

The extract obtained in Example 3 was eluted and dried in the same wayas in Example 6 to obtain 355 mg of kallidinogenase. The kallidinogenaseactivity of this product was 98.5 KU/mg.

What we claim is:
 1. In a method for purifying kallidinogenase bysubjecting the aqueous phase of a kallidinogenase-containing aqueousextract obtained from the pancreas of a mammal to an adsorption-elutiontreatment using an anion exchange resin, the improvement comprising (1)bringing said aqueous phase of the kallidinogenase-containing aqueousextract into contact with a gel-type weakly basic anion-exchange resinto cause the kallidinogenase fraction to be adsorbed to said resin, and(2) eluting the kallidinogenase with an aqueous solution of a saltconsisting of an ammonium salt of a weak acid, thereby to collect thekallidinogenase.
 2. The method of claim 1 wherein said ammonium salt ofa weak acid is selected from the group consisting of ammonium salts ofweak inorganic acid and ammonium salts of fatty acids containing 1 to 5carbon atoms.
 3. The method of claim 2 wherien said ammonium salt isselected from the group consisting of ammonium carbonate, ammoniumbicarbonate, ammonium borate, mono-or di-basic ammonium phosphate,ammonium phoshite, ammonium hypophosphite, ammonium formate, ammoniumacetate, ammonium bimalate, ammonium citrate, ammonium lactate, ammoniumoxalate, ammonium succinate, ammonium tartrate and ammonium valerate. 4.The method of claim 1 in which the adsorption step (1) is carried out ata pH of more than about 4 but below about
 8. 5. The method of claim 1 inwhich the adsorption step (1) is carried out at a pH of from about 5 toabout
 7. 6. The method of claim 1 in which the adsorption step (1) iscarried out at a pH of from about 5.5 to about 6.5.
 7. The method ofclaim 1 in which the elution step (2) is carried out at a pH of fromabout 6 to about
 7. 8. The method of claim 6 in which the elution step(2) is carried out at a pH of from about 6 to about
 7. 9. The method ofclaim 1 in which the gel-type weakly basic anion-exchange resincomprises a resin having secondary or tertiary amine exchange groups.10. The method of claim 8 in which the gel-type weakly basicanion-exchange resin comprises a resin having secondary or tertiaryamine exchange groups.
 11. A method for extracting and collectingpurified kallidinogenase (EC 3.4.4.21 substance) which comprisesextracting the pancreas of a mammal with water in the presence of a saltat a temperature of about 45° to about 60° C. and a pH of about 5 toabout 8, wherein the extraction is carried out in the presence of awater-soluble salt of magnesium with an acid thereby obtaining anaqueous phase containing kallidinogenase, bringing said aqueous phase ofthe kallidinogenase-containing aqueous extract into contact with agel-type weakly basic anion-exchange resin to cause the kallidinogenasefraction to be adsorbed to said resin, and eluting the kallidinogenasewith an aqueous solution of a salt consisting of an ammonium salt of aweak acid, thereby to collect the kallidinogenase.